1. Field of the Invention
The present invention relates to a method for screening a substance that alters cell-stimulating activity using GPR40, a G protein-coupled receptor protein and a phospholipase or a salt thereof, and a screening kit to be used for such screening.
2. Background Art
Many physiologically active substances such as hormones and neurotransmitters regulate biological functions through their specific receptor proteins expressed on cell surface membranes. Many of these receptors share a 7-transmembrane structure which couples with trimeric G protein (guanine nucleotide-binding protein) intracellularly and are accordingly called G-protein coupled receptors (GPCRs).
GPCR is expressed on the cell surface of a variety of functional cells, organs, and organ parts and activates or suppresses cellular functions by transmitting a signal intracellularly via binding to its regulatory molecule. Accordingly, GPCRs play important roles in a variety of organs and organ parts. It is important to clarify interactions between GPCRs and these physiologically active substances for better understanding of biological functions and for the development of drugs that are closely related thereto. The development of these therapeutic drugs requires efficient screening for GPCR agonists and antagonists, functional analyses of a receptor protein expressed in a living body, and expression systems of the gene in appropriate cells.
In recent years, the presence of a number of novel genes have been revealed by a random analysis of cDNA sequences shown in EST database and the like, or a comprehensive analysis of genome DNA. GPCRs share a 7-transmembrane domain and also a number of other common sequences. Because of this, novel members of GPCR have been found among a number of those newly discovered genes. Ligands for these novel GPCRs thus discovered are usually unidentified. Identification of ligands and functional analysis for orphan GPCRs whose ligands are not yet identified are believed to be significantly important because these may provide an opportunity for the development of new therapeutic drugs.
In most cases, it is difficult to predict a ligand for each of orphan GPCRs. Ligands for GPCRs include a wide variety of substances such as biological amines, amino acids, nucleic acids and its metabolites, peptides, proteins (for example, hormones, and chemokines), and lipids. Purification of a ligand from extracts requires an extraction method specific to each type of ligand substances. Also, in general, a type of signal transduction system activated by orphan GPCR after responding to a ligand is not easily predictable and studies are required in miscellaneous expression systems. Since prediction of a tissue in which a ligand is present is not easy, a number of different tissue extracts are required. Thus, the ligand identification for orphan GPCRs faces a great deal of difficulty. Discovery of a novel ligand for GPCR and its direct application, or screening for a new drug using the novel ligand is expected to provide an opportunity to develop new drugs of which action mechanism is novel and entirely different from that of currently available drugs.
GPR40 has been known as one of GPCRs (Biochemical and Biophysiological Research Communications, Vol. 239, pp 543-547, (1997)). Although ligands for GPR40 have not been completely elucidated, fatty acids have been reported as one of the ligands (WO2002/057783 and WO2003/068959).
GPR40 has been known to be expressed in pancreatic cells, Langerhans' islet β cells. GPR40 has been known to be involved in glucose-dependent insulin secretion in a mouse pancreas-derived cell strain MIN6 (WO2003/068959). In addition, GPR40 has been reported to be involved in insulin secretion in primary cultured islet cells (Ito Y et al., Nature, 422 (6928): 173-6, 2003). Further, involvement of GPR40 in proliferation of breast cancer has been reported (Hardy S et al., Journal of Biological Chemistry, 280 (14): 13285-91, 2005). In addition, it has been reported that hyperinsulinemia, fatty liver, hyperglyceridemia and the like induced by obesity are ameliorated in mice with reduced GPR40 expression (Steneberg P et al., Cell Metabolism, 1 (4): 245-58, 2005). Based on these findings, application of GPR40 agonists and antagonists is expected to the treatment of type I diabetes (insulin dependent diabetes), type II diabetes (non-insulin dependent diabetes), diabetic complications and degenerative diseases (for example, diabetic neuropathy, diabetic nephropathy, diabetic retinopathy, and the like), hyperglycemia, polyuria, ketonemia, acidosis, insulin resistance, impaired glucose tolerance, neurodegenerative diseases, insulinoma, cancers, hyperinsulinemia, hyperglyceridemia, fatty liver, hypoglycemia due to insulin hypersecretion, arteriosclerosis, hyperlipidemia, cerebral stroke, obesity, various diseases induced by diabetes or obesity, and the like.
As described above, fatty acids have been reported as ligands for GPR40. However, it is often difficult to prepare a ligand solution with fatty acids since fatty acids are barely soluble in an aqueous solvent system. Also, fatty acids are easily adsorbed to plastics or glass used for screening and unsaturated fatty acids are easily oxidized. Moreover, fatty acids are known to bind to albumin easily. Under physiological conditions, most of fatty acids are bound to blood albumin and only a small part of fatty acids (about 1%) exist as free fatty acids. For that reason, an inhibitory effect of bovine serum albumin (BSA) on GPR40 activity stimulated by fatty add have been observed in the screening of a GPR40 using a fatty acid, and the necessity of screening in the absence of serum or albumin is reported (for example, Ito Y et al., Nature, 422 (6928): 173-6, 2003). On the other hand, generally, the screening for a drug using cells and proteins is often carried out in the presence of serum or albumin (BSA and the like) since conditions closer to physiological conditions are required. If the screening is carried out in the absence of serum, a long time culture is usually difficult because of cellular damages in the serum free condition. It is therefore necessary that cells are cultured in a serum containing medium in advance and the medium must be replaced by a serum free medium at the time of the screening, which makes the process more complicated.
Accordingly, it has been expected for a new screening system not using fatty acid directly to screen for GPR40 agonists or antagonists.
Phospholipase is a family of enzymes that hydrolyze an ester linkage of glycerophospholipids, and is classified into phospholipases A1, A2, B, C, and D depending on the position of the ester linkage to be hydrolyzed. Phospholipase A2 (PLA2) is further classified into secretory (sPLA2), cytoplasmic (cPLA2) and calcium independent (iPLA2) forms. Among them, there are 10 enzymes known for sPLA2.
At the same time, it has been reported that phosphatidylcholine (1-palmitoyl-2-linolenoyl) that has been hydrolyzed by phospholipase A2 and oxidized induces calcium influx in cells expressing G2A, one of GPCRs, and that G2A is activated by an oxidized free fatty acid produced by oxidation and hydrolysis of phosphatidylcholine or cholesteryl linoleate (J. Biol. Chem., Vol. 280, Issue 49, 40676-40683, Dec. 9, 2005).